A major goal of biomedical research is to provide protection against viral disease through immunization. One approach has been to use killed vaccines. However, large quantities of material are required for killed vaccinne in order to retain sufficient antigenic mass. In addition, killed vaccines are often contaminated with undesirable products during their preparation. Heterologous live vaccines, using appropriately engineered adenovirus, which is itself a vaccine, Chanock, R. M. et al., JAMA, 195, 151 (1966), seem an excellent immunogen. Out invention concerns live oral vaccines using adenovirus as vector.
Presently marketed adenovaccine comprises live, infectious adenoviruses in an enteric-coated dosage form. Upon administration to the patient to be vaccinated, the virus is carried past the upper-respiratory system (where disease-producing infection is thought to occur), and is released in the intestine. In the intestine, the virus reproduces in the gut wall, where, although it is not capable of causing adenoviral disease, nevertheless induces the formation of adenovirus antibodies, thus conferring immunity to adenoviral disease. In our invention, live, infectious adenovirus which has been engineered to contain genes coding for antigens produced by other disease-causing organisms is administered in an enteric-coated dosage form. Upon release in the intestine the virus will reproduce in the gut wall, will separately express both the adenoviral antigen and the pathogen surface antigen, and will induce the formation of antibodies or induce cell mediated immunity to both adenovirus and the other disease-causing organism. By "live virus" is meant, in contradistinction to "killed" virus, a virus which is, either by itself or in conjuction with additional genetic material, capable of producing identical progeny. By "infectious" is meant having the capability to deliver the viral genome into cells.
Approximately 200,000 persons in the United States are infected each year with Hepatitis B virus. In addition, there is a strong correlation between hepatitis B infection and liver cancer. The presently marketed vaccines against hepatitis B are injectable products manufactured from the blood plasma of healthy carriers by a lengthy process involving several discrete steps.
There are two major hepatitis B viral antigens: the surface antigen (HB.sub.s Ag) and the core antigen (HB.sub.c Ag). The antigenic structure of HB.sub.s Ag is somewhat complex. There is a common group-specific determinant, a. In addition, there are two sets of mutually exclusive type-specific determinants d or y and w or r. The HB.sub.c Ag is of a single antigenic type. It is known that production of antibody against HB.sub.s Ag or HB.sub.c Ag confers immunity against hepatitis B infection.
Several groups have employed recombinant DNA techniques to synthesize the HB.sub.s Ag by microorganisms. HB.sub.s Ag has been synthesized in Escherichia coli in the form of a fusion protein (Edman, J. C. et al., Nature, 291, 503 (1981)). It has also been synthesized in yeast using the ADH promoter (Valenzuela et al., Nature 298, 347 (1982)) of acid phosphatase promoter (Miyanohara et al., Proc. Natl. Acd. Sci. USA, 80, 1 (1983)). The expression of HB.sub.s Ag by Adenovirus in eukaryotic cell strains has also been described (Perricaudet, et al., European Patent Publication 185,573 (1986)), as has the possibility of using adenoviruses modified at the E3 region by the insertion of recombinant DNA in the constitution of live vaccines (ibid, page 9). Finally, vaccinia virus has been used as a vector to produce a live virus vaccine to hepatitis virus (Smith et al., Nature, 302, 490 (1983)).
Rotaviruses are a major cause of acute gastroenteritis in infants. These viruses possess a genome of eleven double-stranded RNA segments enclosed in a capsid. The capsid contains an inner and outer shell. One of the outer shell proteins, VP7, is a glycoprotein that reacts with serotype-specific neutralizing antibodies (Kalica, A. R. et al., Virology, 112, 385 (1981)). This protein is coded for by gene 9 of the human type 1 (Wa) rotavirus. Gene 9 of type 1 human rotavirus has recently been cloned in E. coli and its sequence determined (Richardson, M. et al., J. Virol, 51, 860 (1984)).
Adenoviruses contain a linear duplex DNA molecule (m.w. 20.times.10.sup.6 -25.times.10.sup.6) that codes for 20-30 polypeptides. Many of these are incorporated into the viral particle which is morphologiclly complex and has a sophisticated assembly process. Previously SV40 T antigen has been expressed using an adenovirus recombinant (Solnick, D. Cell, 24, 135 (1981), Thummel, C. et al., Cell, 23, 825 (1981), Gluzman, Y. et al., in Eukaryotic Viral Vectors, p. 187, Cold Spring Harbor (1982)). Also mouse dihydrofolate reductase has been expressed using an adenovirus recombinant (Berkner, K. and Sharp, P. A., Nucleic Acids Research, 12, 1925 (1984)).
Roy, C., in European Patent Publication 80,806 (1983) proposes a method for producing immunity to microbial diseases by the administration of a microbe containing a foreign gene which will express an antigen of a second microbe to which immunity is desired. He states that preferred oral preparations are enteric-coated. Dulbecco proposes recombinant adenovirus vaccines in which the surface protein of adenovirus is modified to contain in its structure a segment of foreign protein which will produce a desired biological response on administration to animals (PCT International Publication Number WO 83/023 (1983)).